One of the most prevalent bleeding disorders is von Willebrand's disease (VWD), which is caused by missing von Willebrand factor (VWF), reduced levels of VWF or the expression of VWF variants with functional defects.
VWF is one of the largest known plasma proteins. VWF is a multimeric adhesive glycoprotein present in the plasma of mammals, which has multiple physiological functions. During primary hemostasis VWF acts as a mediator between specific receptors on the platelet surface and components of the extracellular matrix such as collagen. Moreover, VWF serves as a carrier and stabilizing protein for procoagulant Factor VIII. VWF is synthesized in endothelial cells and megakaryocytes as a 2813 amino acid precursor molecule. The amino acid sequence and the cDNA sequence of wild-type VWF are disclosed in Collins et al. 1987, Proc Natl. Acad. Sci. USA 84:4393-4397. The precursor polypeptide, pre-pro-VWF, consists of a 22-residue signal peptide, a 741-residue pro-peptide and the 2050-residue polypeptide found in mature plasma VWF (Fischer et al., FEBS Lett. 351: 345-348, 1994). After cleavage of the signal peptide in the endoplasmatic reticulum a C-terminal disulfide bridge is formed between two monomers of VWF. During further transport through the secretory pathway 12 N-linked and 10 O-linked carbohydrate side chains are added. More important, VWF dimers are multimerized via N-terminal disulfide bridges and the propeptide of 741 amino acids length is cleaved off by the enzyme PACE/furin in the late Golgi apparatus. The propeptide as well as the high-molecular-weight multimers of VWF (VWF-HMWM) are stored in the Weibel-Pallade bodies of endothelial cells or in the α-Granules of platelets.
Once secreted into plasma the protease ADAMTS13 cleaves VWF within the A1 domain of VWF. Plasma VWF therefore consists of a whole range of multimers ranging from single dimers of 500 kDa to multimers consisting of up to more than 20 dimers of a molecular weight of over 10,000 kDa. The VWF-HMWM hereby having the strongest hemostatic activity, which can be measured in ristocetin cofactor activity (VWF:RCo). The higher the ratio of VWF:RCo/VWF antigen, the higher the relative amount of high molecular weight multimers.
Defects in VWF are causal to VWD 0, which is characterized by a more or less pronounced bleeding phenotype. VWD type 3 is the most severe form in which VWF is completely missing, VWD type 1 relates to a quantitative loss of VWF and its phenotype can be very mild. VWD type 2 relates to qualitative defects of VWF and can be as severe as VWD type 3. VWD type 2 has many sub forms some of them being associated with the loss or the decrease of high molecular weight multimers. Von VWD type 2a is characterized by a loss of both intermediate and large multimers. VWD type 2B is characterized by a loss of highest-molecular-weight multimers.
VWD is the most frequently inherited bleeding disorder in humans and can be treated by replacement therapy with concentrates containing VWF of plasmatic or recombinant origin. VWF can be prepared from human plasma as for example described in EP 0503991. EP 0784632 describes a method for isolating recombinant VWF.
In plasma Factor VIII binds with high affinity to von VWF, which binding protects Factor VIII from premature catabolism and VWF thus plays in addition to its role in primary hemostasis a crucial role to regulate plasma levels of Factor VIII and as a consequence is also a central factor to control secondary hemostasis.
Recombinant or plasma derived VWF or VWF/Factor VIII complexes are used to provide pharmaceutical preparations to treat VWD and VWF/Factor VIII complexes have been also used to treat the medical indication hemophilia A in which disease Factor VIII is either missing or only available in reduced concentrations or as a Factor VIII variant with reduced functionality.
In order to provide such pharmaceutical preparations there is a need for efficient and scalable purification methods for VWF.
Due to its enormous size VWF multimers are especially sensitive to shear stress. Already at a sheer stress of above 2000 sec−1 VWF multimers begin to unfold and the unfolded VWF is then prone to degradation and denaturation. Common methods to concentrate therapeutic proteins in industrial manufacturing processes like ultrafiltration are not suitable to concentrate VWF in industrial scale manufacturing because they exert too high sheer stress. WO 2010/025278 offers one technical solution to achieve the concentration of VWF without compromising its specific activity using hollow fibre membranes, but there is a need to identify alternative technologies to concentrate VWF from solutions comprising VWF which are simpler, cheaper and scalable while preserving the structural integrity and biological activity of this complex macromolecule.
Precipitation of proteins is a technique known in the prior art. However, for each given protein the optimal precipitation agent and precipitation parameters must be identified. One way how precipitation can be achieved is by salting out procedures (for example by using ammonium sulphate) where the ionic components of the salt used for the precipitation compete with the target protein for water molecules and if the concentration of the salt ions is high enough, the target protein becomes insufficiently hydrated to be kept in solution leading to the precipitation of the target protein. However precipitates obtained by salting out procedures are often difficult to resolubilize. Therefore a need exists to find methods to precipitate VWF which allow an easy and gentle resolubilization, while preserving the structural integrity and biological activity of VWF.
Also precipitation is often carried out as a co-precipitation, where the precipitating agent also precipitates with the protein of interest. Depending on the nature of the precipitating agent the target protein must then be purified from the precipitating agent, such that the target protein is not contaminated by the precipitating agent. Powerful precipitating agents like synthetic polyelectrolytes can be very difficult or slow to remove and release from the target protein.
Precipitation of vWF with a combination of glycine and sodium chloride have also been described in the prior art (EP1405863). However, high amounts of salts are needed and it is difficult to avoid the unwanted precipitation of other components from the solution than the target protein. If for example the VWF solution still contains a surfactant like pluronic acid which commonly used in cell culture media, precipitation conditions as described in EP1405863 may lead to the co-precipitation of pluronic acid which may even comprise the precipitation of the target protein.
U.S. Pat. No. 5,679,776 discloses the use of 80 mM barium chloride in order to remove prothrombin complex from a solution of VWF complexed to Factor VIII by way of precipitating the prothrombin complex but not the VWF/Factor VIII complex. Surprisingly it was now found that a combination of calcium ions—which is like barium an alkaline earth metal—with phosphate ions can be used to precipitate VWF in high yield, which precipitate can then be resolubilized under gentle conditions with a calcium complexing agent while retaining the biological activity of VWF.